Advances in nuclear transfer and embryonic stem cell technology have facilitated the cloning of non-human animals for diverse applications including agriculture, xenotransplantation, disease models, recombinant protein production, and novel means of manufacturing human cells for use in medical therapies, diagnosis, and discovery research. Each of these practical applications would benefit from new technologies to improve efficiencies in the production of animals, tissues, and cells. In the case of animal cloning, the high cost of recipient females to gestate the cloned fetuses often makes the commercialization of cloned animals impractical. In the case of the therapeutic uses of pluripotent stem cells, many pluripotent cells such as human embryonic stem (hES) cells, are problematic to culture using traditional cell culture technology. The cells are dependent on a close association with similar undifferentiated cells and often require being cultured in juxtaposition with embryonic fibroblast feeder cells in order to maintain them in the undifferentiated state.
In addition, while some cells such as hES cells have a demonstrated potential to differentiate into any and all of the cell types in the human body including complex tissues, and while genes expressed uniquely in many differentiated cell types are known allowing genetic selection and purification of populations of any cell type of interest, nevertheless, there is need for new technologies to influence the differentiation of pluripotent stem cells such as hES cells, new means of allowing the cells to differentiate in a three dimensional tissue culture environment, and novel means of purifying the target cells of interest, and techniques such as these that can be performed in SPF conditions to minimize the risk of pathogen transmission into humans.
In the field of the cultivation of human cells for human cell therapy, regulatory agencies require production methods wherein the cells are grown in defined conditions with stringent control over contact of the cells (or anything that may come in contact with the cells) with uncharacterized materials that are a potential source of pathogens. In the case of human embryonic stem (hES) cells, it is desirable to identify a means of cultivating the cells in pathogen-free conditions, differentiating downstream progeny of the cells, scale up the number of the cells for batch production, cryopreservation, and genetic modification.
The original culture of hES cells as reported by Thomson et al (Science. 1998 Nov. 6; 282(5391):1145-7) was accomplished by culturing the inner cell mass of human blastocysts in co-culture with feeder layer of embryonic murine fibroblasts under culture conditions well known in the art of tissue culture to generate ES cell lines. The murine fibroblasts provide largely uncharacterized factors that promote the growth of ES cells while maintaining them in an undifferentiated state. However, the embryonic murine fibroblasts are also a potential source of pathogens including uncharacterized retroviruses. Therefore, novel means of isolating, culturing, and differentiating hES cells and other cells are of great practical value. While avian CEFs have been shown to support the growth of murine ES cells (Yang & Petitte, 1994), and the use of avian cytokines has been described in in non-human mammalian embryonic stem cell culture, (Poultry Science 73: 965-974), there has been no description of the possibility that avian CEFs could be useful in providing SPF support for the growth of other mammalian ES cells such as hES cells.
In addition, because of the innate capacity of hES cells to organize into complex three dimensional tissues including organogenesis, and because the growth of tissues in culture systems beyond the size of approximately 0.5 mm in thickness is impractical without a means of supplying vascular support, there is a need for developing conditions that allow for the growth of solid tissues and conditions that provide suitable vascular support for such growing tissue with a dimensions of greater than 0.5 mm while maintaining the cells in a specific pathogen-free environment.
The avian egg is a relatively well-characterized structure that has evolved as a means of providing physiological support to a developing vertebrate embryo, including nutritional support, waste disposal, and gas exchange. The ovum of avian species such as the domestic chicken (Gallus domesticus) is that part of the egg commonly called the “yolk” (FIG. 1). The bulk of the ovum is a colloidal suspension of nutrients while a small volume of cytoplasm is concentrated in a region approximately 3 mm in diameter called the blastodisc on the animal pole. Following fertilization, the ovum traverses the oviduct acquiring albuminous material (egg white) and finally the shell membrane and the calcified egg shell.
In the case of an egg that has become fertilized by sperm subsequent to ovulation and prior to encapsulation into the shell, the blastodisc will undergo repeated rounds of karyokinesis and cytokinesis until at about the time the egg is laid, a collection of cells called the blastoderm has formed that is roughly equivalent to the stage of mammalian embryos at the blastocyst stage. Therefore, cultured avian blastodermal cells are occasionally referred to as avian embryonic stem cells (aES cells) and those from species of domestic chicken are referred to as chicken embryonic stem (cES) cells (U.S. Pat. No. 5,340,740). Following the formation of primitive germ layers of the avian embryo proper, extraembryonic membranes begin to form that will function to support the developing embryo. As shown in FIG. 2, these include the splanchnopleure that will form the yolk sac, the somatopleure, that will form the amnion and the chorion, and the allantoic membrane, that will eventually fuse with the chorion to form the chorioallantoic membrane. These membranes become vascularized and provide the developing embryo with nutrients from the yolk sac and gas exchange across the egg shell.
In contrast to avian species, mammalian development is viviparous and often occurs in the context of the uterus, where embryonic membranes form analogous to that in the avian egg, but the extraction of nutrition from the maternal circulation can occur either through either the chorion, the allantoic membrane, or the yolk sac membrane depending on the mammalian species. Generally speaking, in most mammals, the yolk sac provides little if any nutritional support.
The avian egg provides an unusually promising environment for the cultivation of human cells. As described herein, novel means of culturing and maintaining hES cells, hED cells, and cells differentiated from such cells are described utilizing telolecithal or eutelolecithal eggs or cells derived from embryonated telolecithal or eutelolecithal eggs. In addition, it is possible to utilize telolecithal or eutelolecithal eggs to support the in ovo development on non-human mammalian embryos and fetuses and to reconstitute embryonic stem cells and embryo-derived cells from chromatin from mammalian species.